2,3-dihydro-isoindole-1-on derivative as BTK kinase suppressant, and pharmaceutical composition including same

ABSTRACT

The present invention provides a compound selected from the group consisting of a compound of formula (I), pharmaceutically acceptable salts, esters, prodrugs, hydrates, solvates and isomers thereof; a use of the compound for the treatment, relief or prevention of diseases caused by abnormal or uncontrolled activation of protein kinase, and a use of the compound for the manufacture of a medicament for the treatment, relief or prevention of the diseases; a pharmaceutical composition comprising the compound as an active ingredient; and a method for the treatment, relief or prevention of the diseases using the compound. The inventive compound is useful for the treatment, relief or prevention of diseases caused by abnormal or uncontrolled activation of protein kinase.

This application is a national phase filing of PCT Application No.PCT/KR2013/012204 to HONG et al., filed Dec. 26, 2013, entitled“2,3-Dihydro-Isoindole-1-On Derivative as BTK Kinase Suppressant, andPharmaceutical Composition Including Same,” which claims priority toU.S. Provisional Application No. 61/746,980 filed Dec. 28, 2012,entitled “2-3-Dihydro-Isoindol-1-One Derivatives and Methods of UseThereof as BTK Inhibitor”.

FIELD OF THE INVENTION

The present invention relates to a compound selected from the groupconsisting of a compound of formula (I) and pharmaceutically acceptablesalts, esters, prodrugs, hydrates, solvates and isomers thereof, and apharmaceutical composition comprising same as an active ingredient forthe treatment, relief or prevention of diseases caused by abnormal oruncontrolled activation or protein kinase.

BACKGROUND OF THE INVENTION

Bruton's tyrosine kinase (BTK) is a member of the TEC family ofnonreceptor tyrosine kinases, which consists of 650 amino acid residuesand contains pleckstrin homology (PH) domain, zinc-finger region, SH3domain, SH2 domain, and kinase domain. Recently, kinase domain, amongsaid domains, is gaining more interests as a drug target.

BTK is found in B-cells and hematopoietic cells, rather than someT-cells, natural killer cells, plasma cells, etc. When BTK is stimulatedby the B-cell membrane receptor (BCR) signals that are caused by variousinflammatory responses or cancers, BTK plays an important role inproduction of cytokines such as TNF-α, IL-6, etc., as well as NF-κB byinitiating downstream signaling such as phospholipase C gamma 2 (PLCγ2).

In the treatment of inflammation, BTK is known for mediating responsesof the membrane receptors, e.g., B-cell antigen receptors which detectinflammation-inducing substances, CD40, TLR-4, Fcg and the like. Also,BTK has a strong influence on the signaling mechanism of inflammationcaused by stimulation of mast cell, B-cell and macrophage. Therefore,inhibition of BTK may block IgE signaling which may slow down theprogression of diseases caused by abnormal activation of BTK. Thissignaling mechanism is a complicated signaling pathway ofimmunosubstance secretion. In this process, protein phosphorylation anddephosphorylation take place in a multi-step procedure, and since BTK isone of the high-level steps in the signaling pathway, along with spleentyrosine kinase (SYK) and, thus, it is more effective for preventingactivation of factors that cause immune responses than other kinasetargets.

Further, in the treatment of cancer, it is known that BTK modifies BCRand B-cell surface proteins which generate antisuicide signals. Thus,inhibition of BTK may bring about anticancer effects against cancersthat are associated with BCR signaling such as lymphoma. In fact,Ibrutinib (PCI-32765) developed by Pharmacyclics Inc. was recentlyapproved as an anti-cancer agent for the treatment of chroniclymphocytic leukemia (CLL), and a phase III trial of AVL-292 developedby Avila Therapeutics for small lymphocytic leukemia (SLL) and CLL iscurrently underway. It has been proven that these compounds are quiteeffective against SLL and CLL that are relatively rare type of cancers.However, they have failed to achieve satisfactory results againstdiffuse large B-cell lymphoma (DLBCL) which is more prevalent type oflymphoma. Thus, there is a growing demand for a noble drug.

The action mechanism of BTK inhibitor as an anti-inflammatory agent aswell as an anti-cancer agent is thoroughly described in the reference[Nature Chemical Biology 7, (2011), 4].

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide acompound selected from the group consisting of a compound of formula(I), pharmaceutically acceptable salts, esters, prodrugs, hydrates,solvates and isomers thereof.

It is another object of the present invention to provide apharmaceutical composition comprising same as an active ingredient forthe treatment, relief or prevention of diseases caused by abnormal oruncontrolled activation of protein kinase.

In accordance with an aspect of the present invention, there is provideda compound selected from the group consisting of a compound of formula(I) below and pharmaceutically acceptable salts, esters, prodrugs,hydrates, solvates and isomers thereof:

wherein,

A is

and R₈ is hydrogen, halogen, or C₁₋₃alkyl,

R₁ and R₂ are each independently hydrogen or C₁₋₃alkyl,

R₃ to R₇ are each independently hydrogen, halogen, cyano, nitro orC₁₋₃haloalkyl.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical composition comprising same as an activeingredient for the treatment, relief or prevention of diseases caused byabnormal or uncontrolled activation of protein kinase.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention are explained in detailhereinafter.

In the present invention, there is provided a compound selected from thegroup consisting of a compound of formula (I) below and pharmaceuticallyacceptable salts, esters, prodrugs, hydrates, solvates and isomersthereof:

wherein,

A is

and R₈ is hydrogen, halogen, or C₁₋₃alkyl,

R₁ and R₂ are each independently hydrogen or C₁₋₃alkyl,

R₃ to R₇ are each independently hydrogen or electron withdrawingsubstituent, wherein the electron withdrawing substituent is, forexample, halogen, cyano, nitro or C₁₋₃haloalkyl.

In one specific embodiment, said R₃ to R₇ are each independentlyhydrogen, fluoro, chloro, bromo, iodo, cyano, nitro, difluoromethyl ortrifluoromethyl.

In another specific embodiment,

said R₁ and R₂ are each independently hydrogen or methyl;

R₃ to R₇ are each independently hydrogen, fluoro, chloro, cyano ortrifluoromethyl;

R₈ is hydrogen or fluoro.

The term “halo” or “halogen” as used herein refers to fluorine,chlorine, bromine or iodine, unless otherwise indicated.

The term “alkyl” as used herein refers to linear or branched hydrocarbonresidues, unless otherwise indicated.

The compound of formula (I) according to the present invention may forma pharmaceutically acceptable salt derived from inorganic or organicacid, and such salt may be pharmaceutically acceptable nontoxic acidaddition salt containing anion. For example, the salt may include acidaddition salts formed by inorganic acids such as hydrochloric acid,sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid,hydroiodic acid, and the like; organic carbonic acids such as tartaricacid, formic acid, citric acid, acetic acid, trichloroacetic acid,trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaricacid, maleic acid, and the like; and sulfonic acids such asmethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,naphthalensulfonic acid, and the like. Among them, acid addition saltsformed by sulfuric acid, methanesulfonic acid or hydrohalogenic acid andthe like are preferred.

The “pharmaceutically acceptable salt” of the compound of formula (I)may be prepared by conventional methods well-known in the art.Specifically, the “pharmaceutically acceptable salt” in accordance ofthe present invention may be prepared by, e.g., dissolving the compoundof formula (I) in a water-miscible organic solvent such as acetone,methanol, ethanol or acetonitrile and the like; adding an excessiveamount of organic acid or an aqueous solution of inorganic acid thereto;precipitating or crystallizing the mixture thus obtained. Further, itmay be prepared by further evaporating the solvent or excessive acidtherefrom; and then drying the mixture or filtering the extract by usinga suction filter.

The term “ester” as used herein refers to a chemical moiety havingchemical structure of —(R)n-COOR′, wherein R and R′ are eachindependently selected from the group consisting of alkyl, cycloalkyl,aryl, heteroaryl (connected to oxygen atom by aromatic ring) andheteroalicyclic (connected by aromatic ring), and n is 0 or 1, unlessotherwise indicated.

The term “prodrug” as used herein refers to a precursor compound thatwill undergo metabolic activation in vivo to produce the parent drug.Prodrugs are often useful because they can be easily administered ascompared to parent drugs thereof in some cases. For instance, someprodrugs are bioavailable via oral administration unlike parent drugsthereof often show poor bioavailability. Further, the prodrugs may showimproved solubility in the pharmaceutical composition as compared toparent drugs thereof. For instance, the compound of formula (I) may beadministered in the form of an ester prodrug so as to increase drugdelivery efficiency since the solubility of a drug can adversely affectthe permeability across the cell membrane. Then, once the compound inthe form of the ester prodrug enters a target cell, it may bemetabolically hydrolyzed into a carboxylic acid and an active entity.

Hydrates or solvates of the compound of formula (I) are included withinthe scope of the present invention.

Further, the compound of formula (I) of the present invention can havean asymmetric carbon center, and thus may be present in the form ofisomer including enantiomer, diastereomer or racemic mixture, suchentire stereoisomers and mixtures being included within the scope of thepresent invention.

Particular examples of the compound of formula (I) of the presentinvention are as follow:

-   1)    1-(2,6-dichloro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   2)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-trifluoromethyl-phenyl)-urea;-   3)    1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   4)    1-(2-chloro-6-fluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   5)    1-(2,6-bis-trifluoromethyl-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   6)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-fluoro-6-trifluoromethyl-phenyl)-urea;-   7)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-    urea;-   8)    1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-1-methyl-urea;-   9)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-pentafluorophenyl-urea;-   10)    1-(2,5-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)pheny)urea;-   11)    1-(2,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   12)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,6-trifluoro-phenyl)-urea;-   13)    1-(3,5-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   14)    1-(3,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;-   15)    1-(4-cyano-3-fluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;-   16)    1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;-   17)    1-(3-chloro-2,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)pheny)urea;-   18)    1-(2-chloro-3,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)pheny)urea;-   19)    1-(4-chloro-2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;    and-   20)    1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,5,6-tetrafluoro-phenyl)-urea.

The compound selected from the group consisting of the compound offormula (I), pharmaceutically acceptable salts, esters, prodrugs,hydrates, solvates and isomers thereof may be used for the treatment,relief or prevention of diseases caused by abnormal or uncontrolledactivation of protein kinase such as ABL (Abelson tyrosine kinase), ACK(Activated cdc42-associated kinase), AXL, Aurora, BLK (B lymphoidtyrosine kinase), BMX (Bone marrow X-linked kinase), BTK (Bruton'styrosine kinase), CDK (Cyclin-dependent kinase), CSK (C-Src kinase), DDR(Discoidin domain receptor), EPHA (Ephrin type A receptor kinase), FER(Fer (fps/fes related) tyrosine kinase), FES (Feline sarcoma oncogene),FGFR (Fibroblast growth factor receptor), FGR, FLT (Fms-like tyrosinekinase), FRK (Fyn-related kinase), FYN, HCK (Hemopoietic cell kinase),IRR (Insulin-receptor-related-receptor), ITK (Interleukin 2-inducible Tcell kinase), JAK (Janus kinase), KDR (Kinase insert domain receptor),KIT, LCK (Lymphocyte-specific protein tyrosine kinase), LYN, MAPK(Mitogen activated protein kinase), MER (c-Mer proto-oncogene tyrosinekinase), MET, MINK (Misshapen-like kinase), MNK (MAPK-interactingkinase), MST (Mammalian sterile 20-like kinase), MUSK (Muscle-specifickinase), PDGFR (Platelet-derived growth factor receptor), PLK (Polo-likekinase), RET (Rearranged during transfection), RON, SRC (Steroidreceptor coactivator), SRM (Spermidine synthase), TIE (Tyrosine kinasewith immunoglobulin and EGF repeats), SYK (Spleen tyrosine kinase), TNK1(Tyrosine kinase, non-receptor, 1), TRK (Tropomyosin-receptor-kinase),TNIK (TRAF2 and NCK interacting kinase) and the like.

Accordingly, the present invention provides a use of the compound offormula (I), pharmaceutically acceptable salts, esters, prodrugs,hydrates, solvates and isomers thereof for the treatment, relief orprevention of diseases caused by abnormal or uncontrolled activation ofprotein kinase, and a use of the compound for the manufacture of amedicament for the treatment, relief or prevention of the diseases.

Further, the present invention provides a method for the treatment,relief or prevention of diseases caused by abnormal or uncontrolledactivation of protein kinase in a mammal, comprising administering tothe mammal a pharmaceutical composition comprising the compound offormula (I), pharmaceutically acceptable salts, esters, prodrugs,hydrates, solvates and isomers thereof.

Furthermore, the present invention provides a pharmaceutical compositionfor the treatment, relief or prevention of diseases caused by abnormalor uncontrolled activation of protein kinase in a mammal, comprising thecompound of formula (I), pharmaceutically acceptable salts, esters,prodrugs, hydrates, solvates and isomers thereof.

The said diseases related with kinase activity may include any diseasecaused by abnormal or uncontrolled activation of protein kinase.Specific examples thereof may be cancer, inflammation associated withrheumatoid arthritis and osteoarthritis, asthma, allergy, atopicdermatitis, or psoriasis, but not limited hereto.

Examples of said cancer include lymphoma, leukemia, blood cancer,stomach cancer, non-small cell lung cancer, liver cancer, colorectalcancer, small bowel cancer, pancreatic cancer, brain cancer, bonecancer, melanoma, breast cancer, sclerosing adenosis, uterine cancer,cervical cancer, ovarian cancer, head and neck cancer, esophagealcancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma,prostate cancer, urethral cancer, bladder cancer, fibroadenoma, orglioblastoma, but not limited hereto.

The pharmaceutical composition may further comprise at least oneadditive selected from the group consisting of antibiotic, alkylatingagent, antimetabolite, hormonal agent, immunological agent,interferon-type agent and anticancer agent.

The pharmaceutical composition of the present invention may beformulated directly, or further contain conventional non-toxicpharmaceutically acceptable additives, e.g., a carrier and an excipient,to be formulated in accordance with any of the conventional methodswell-known in the art.

The method for the treatment, relief or prevention may include, e.g.,administering an effective amount of the pharmaceutical compositioncomprising the compound of the present invention to a subject sufferingfrom, or at risk of, chronic renal failure, diabetes, cancer, AIDS,radiation therapy, chemotherapy, kidney dialysis or anemia caused bysurgery. In one embodiment, the subject is preferably mammal, and morepreferably, human.

The effective amount of the pharmaceutical composition of the presentinvention may be determined by conducting an ordinary test in order tofind out the most effective administration route and suitablepreparation method. The pharmaceutical composition of the presentinvention may be prepared into any type of formulation and drug deliverysystem by using any of the conventional methods well-known in the art.The inventive pharmaceutical composition may be formulated intoinjectable formulations, which may be administereby by routes includingintrathecal, intraventricular, intravenous, intraperitoneal, intranasal,intraocular, intramuscular, subcutaneous or intraosseous. Also, it mayalso be administered orally, or parenterally through the rectum, theintestines or the mucous membrane in the nasal cavity (see Gennaro, A.R., ed. (1995) Remington's Pharmaceutical Sciences). Preferably, thecomposition is administered topically, instead of enterally. Forinstance, the composition may be injected, or delivered via a targeteddrug delivery system such as a reservoir formulation or a sustainedrelease formulation.

The pharmaceutical formulation of the present invention may be preparedby any well-known methods in the art, such as mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping, or lyophilizing processes. As mentioned above, thecompositions of the present invention may include one or morephysiologically acceptable carriers such as excipients and adjuvantsthat facilitate processing of active molecules into preparations forpharmaceutical use.

Proper formulation is dependent upon the route of administration chosen.For injection, for example, the composition may be formulated in anaqueous solution, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In a preferred embodiment of the presentinvention, the inventive compound may be prepared in an oralformulation. For oral administration, the compounds can be formulatedreadily by combining the active compounds with pharmaceuticallyacceptable carriers known in the art. Such carriers enable the inventivecompounds to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject. The compounds may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical preparations for oral use may be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable adjuvants, if desired, toobtain tablets or dragee cores. Suitable excipients may be, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose formulation such as maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and/or polyvinylpyrrolidone (PVP) formulation. Also, disintegratingagents may be employed, such as cross-linked polyvinylpyrrolidone, agar,or alginic acid or a salt thereof such as sodium alginate. Also, wettingagents, such as sodium dodecyl sulfate and the like, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compounds doses.

Pharmaceutical formulations for oral administration may include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In one embodiment, the compounds of the present invention may beadministered transdermally, such as through a skin patch, or topically.In one aspect, the transdermal or topical formulations of the presentinvention can additionally comprise one or multiple penetrationenhancers or other effectors, including agents that enhance migration ofthe delivered compound. Preferably, transdermal or topicaladministration may be used, e.g., in situations in which locationspecific delivery is desired.

For administration by inhalation, the compounds of the present inventionmay be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, e.g., gelatin, foruse in an inhaler or insufflators may be formulated. These typicallycontain a powder mix of the compound and a suitable powder base such aslactose or starch. Compositions formulated for parenteral administrationby injection, e.g., by bolus injection or continuous infusion, can bepresented in unit dosage form e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Formulations for parenteraladministration include aqueous solutions or other compositions inwater-soluble form.

Suspensions of the active compounds may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehicles mayinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes.

Aqueous injection suspensions may contain substances that increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions of the present invention may alsobe formulated as a reservoir formulation. Such long acting formulationsmay be administered by implantation (e.g., subcutaneous orintramuscular) or by intramuscular injection. Thus, for example, theinventive compounds may be formulated with suitable polymeric orhydrophobic materials (e.g., an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, e.g., a sparinglysoluble salt.

For any composition used in the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well-known in the art. For example, based oninformation obtained from a cell culture assay, a dose can be formulatedin animal models to achieve a circulating concentration range thatincludes the IC₅₀. Similarly, dosage ranges appropriate for humansubjects can be determined, for example, using data obtained from cellculture assays and other animal studies.

A therapeutically effective dose of an agent refers to the amount of theagent that results in amelioration of symptoms or a prolongation ofsurvival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, for example, by determining theLD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, whichcan be expressed as the ratio LD₅₀/ED₅₀. Agents that exhibit hightherapeutic indices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methodswell-known in the art, in view of the specifics of a subject'scondition.

In addition, the amount of agent or composition administered will bedependent on a variety of factors, including the age, weight, sex,health condition, degree of disease of the subject being treated, theseverity of the affliction, the manner of administration, and thejudgment of the prescribing physician.

Hereinafter, an exemplary method for preparing the compound of thepresent invention is explained.

Various starting materials may be prepared in accordance withconventional synthetic methods well-known in the art. Some of thestarting materials are commercially available from manufacturers andsuppliers of reagents, such as Aldrich, Sigma, TCI, Wako, Kanto,Fluorchem, Acros, Abocado, Alfa, Fluka, etc., but not limited thereto.

The compounds of the present invention can be prepared from readilyavailable starting materials by conventional methods and processesbelow. Different methods may also be used for manufacturing theinventive compounds, unless otherwise specified as typical or preferredprocess conditions (i.e., reaction temperature, time, molar ratio ofreactants, solvents, pressures, etc.). The optimal reaction conditionsmay vary depending on the particular reactants or solvents employed.Such conditions, however, can be determined by the skilled in the art byconventional optimization process.

In addition, those of ordinary skill in the art recognize that somefunctional groups can be protected/deprotected using various protectinggroups before a certain reaction takes place. Suitable conditions forprotecting and/or deprotecting specific functional group, and the use ofprotecting groups are well-known in the art.

For example, various kinds of protecting groups are described in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Secondedition, Wiley, New York, 1991, and other references cited above.

In one embodiment of the present invention, the compound of formula (I)of the present invention may be prepared by synthesizing anintermediate, Compound D, according to the Reaction Scheme 1 as shownbelow, and then subjecting Compound D through the procedure of ReactionScheme 2 or 3. However, the method for synthesizing Compound D above isnot limited to Reaction Scheme 1.

The method for preparing the starting material of Reaction Scheme 1,i.e., Compound A, is described in International Patent PublicationWO2012/014017, and Compound D is prepared by the following methods.

<1-1> Synthesis of Compound B

Compound A (40 g, 168 mmol) was dispersed in acetic acid (400 mL), addedwith water (400 mL) and pyridine (800 mL), and then the temperature ofthe mixture thus formed was lowered to 10° C. The mixture was added withsodium phosphate monobasic monohydrate (280 g, 2.01 mol), and thenfurther added with Raney Ni (101 g) in water (70 mL) to form a reactionsolution. The reaction solution was heated to 50° C., allowed to reactfor 2 hours. Upon the completion of the reaction, the solution wascooled and filtered. The solution was washed with ethyl acetate (EA, 2.5L), and the filtrate was added with water (800 mL) for extraction. Anorganic layer thus formed was separated, and concentrated under reducedpressure. Cooled water (800 mL) was added thereto, and a solid thusobtained was filtered and dried to obtain Compound B (26.7 g, yield:66%).

¹H-NMR Spectrum(300 MHz, DMSO-d₆): 11.10 (s, 1H), 9.15 (s, 1H), 7.95 (d,J=8.1 Hz, 1H), 7.78 (d, J=8.1 Hz, 1H), 4.41 (s, 2H)

LCMS [M+1]: 241.1

<1-2> Synthesis of Compound C

Compound B (26.7 g, 111 mmol) was dispersed in ethanol (800 mL), andaqueous solutions of 48% methyl glyoxal (67 mL) and 28% ammonia (75 mL)were added thereto. The reaction solution thus formed was heated to 90°C., and stirred for 3 hours. Upon the completion of the reaction, thesolution was concentrated under reduced pressure to reduce the volume ofthe reaction solution to about 200 mL, and a solid thus formed wasfiltered. The solid was washed with ethanol (50 mL) to obtain Compound C(17.2 g, yield: 53%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.21-14.12 (m, 1H), 9.48 (s, 1H),8.27 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.4 Hz, 1H), 7.07-6.82 (m, 1H), 4.39(s, 2H), 2.27-2.18 (m, 3H)

LCMS [M+1]: 293.1

<1-3> Synthesis of Compound D

Compound C (17.2 g, 58.9 mmol),3-fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine(19.5 g, 82 mmol), LiCl (6.9 g, 165 mmol) and Pd(PPh₃)₄ (6.8 g, 5.9mmol) were dispersed in a mixed solution of toluene (589 mL) and ethanol(589 mL), added with an aqueous solution of 1 N Na₂CO₃ (117 mL), andallowed to react at 85° C. for 12 hours. Upon completion of thereaction, the reaction solution was completely concentrated underreduced pressure. A mixed solution of acetone (1.2 L) and acetonitrile(1.2 L) was added thereto, and the reaction solution was stirred for 2hours at 80° C., cooled, filtered and then washed with acetonitrile (0.5L). The filtered solution was concentrated under reduced pressure toreduce the volume of the reaction solution to about 150 mL, and thenfiltered. A solid thus obtained was washed under reduced pressure withacetonitrile (60 mL), n-hexane (100 mL), and water (100 mL),respectively, and dried to yield Compound D,4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(13.31 g, yield: 70%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.44-14.34 (m, 1H), 9.36 (s, 1H),8.36 (d, J=8.4 Hz, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.16 (t, J=8.7 Hz, 1H),7.04-6.79 (m, 1H), 6.49-6.42 (m, 2H), 5.65 (s, 2H), 4.36 (s, 2H),2.28-2.18 (m, 3H)

LCMS [M+1]: 323.3

In order to prepare various compounds which can be represented byformula (I) of the present invention, a method for synthesizing suchcompounds by using the intermediate Compound D is specifically describedin Reaction Schemes 2 and 3 below. However, this is a representativeexample of preparing the compound of formula (I) using the intermediateCompound D, and therefore, the preparation method of the compound offormula (I) is not limited hereto.

Compound E disclosed in Reaction Scheme 2 was prepared by the followingmethods.

<2-1> Synthesis of Compound E According to Reaction Scheme 2

4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 1 equivalent) was dispersed in DMF/THF (1:4) to form asolution (0.08 m), and then pyridine (1.15 equivalent) and 4-nitrophenylcarbonochloridate (1.15 equivalent) were added thereto, followed bystirring for 4 hours. After the reaction had reached completion (TLC),n-hexane (same volume as THF, the reaction solution) was added thereto,followed by stirring for 30 minutes. A solid thus formed was washed witha mixed solvent of n-hexane:THF=1:1 (four times the volume of THF,reaction solution), filtered, and then dried. The dried compound wasdispersed in DMF to form a solution (0.1 m), added with substitutedphenylamine (6 to 15 equivalents), and then stirred for 20 minutes undermicrowave conditions (250 W, 250 psi, 150° C.). The reaction solutionwas diluted with ethyl acetate containing 5% methanol, and then washedwith a saturated aqueous solution of NaHCO₃ and water. The organic layerwas dried over anhydrous magnesium sulfate (MgSO₄), and then purified bysilica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain Compound E.

Compound E obtained in Reaction Scheme 2 may also be synthesized byReaction Scheme 3 below.

<3-1> (A) Synthesis of Compound E According to Reaction Scheme 3

Substituted benzoic acid (2 equivalents) was dispersed in diethyl etherto form a mixture (0.08 m), added with phosphorus pentachloride (PCl₅,2.2 equivalents), and then stirred for 2 hours. Upon completion of thereaction, the organic solvent was concentrated under reduced pressurebelow room temperature, and then the reaction solution was diluted (0.08m) by adding acetone to the reactant. Subsequently, sodium azide (NaN₃,2.4 equivalents) in water ( 1/12 volume of acetone) was slowly added tothe reaction solution dropwise at 0° C. After stirring for 2 hours atroom temperature, the reactant was diluted with ethyl acetate, and thenwith water. The organic layer was dried over anhydrous magnesium sulfate(MgSO₄), dispersed in THF to form a solution (0.04 m), added with4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 1 equivalent), and stirred for 4 hour at 90° C. Uponcompletion of the reaction, the solvent was concentrated under reducedpressure, and then purified by silica gel column chromatography (eluent:methylene chloride:methanol=20:1) to obtain Compound E.

Compound E synthesized in Reaction Scheme 2 above may also besynthesized by using another method according to Reaction Scheme3.

<3-2> (B) Synthesis of Compound E According to Reaction Scheme 3

Substituted benzoic acid (2 equivalents) was dispersed in THF to form asolution (0.05 m), and then triethylamine (4 equivalents) anddiphenylphosphorazidate (DPPA, 2.3 equivalents) were added thereto,followed by stirring for 2 hours at room temperature. The reactionsolution was added with4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 1 equivalent), and then stirred for 4 hours at 90° C.Subsequently, the reaction solution was diluted with ethyl acetatecontaining 5% methanol, and washed with water and a saturated aqueoussolution of NaHCO₃. The organic layer was dried over anhydrous magnesiumsulfate (MgSO₄), and then concentrated under reduced pressure. Theconcentrate thus obtained was purified by silica gel columnchromatography (eluent: methylene chloride:methanol=20:1) to obtainCompound E.

Hereinafter, the present invention is described more specifically by thefollowing Examples, but these are provided for illustration purposesonly, and the present invention is not limited thereto.

Example 1: Preparation of1-(2,6-dichloro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.1 g, 0.31 mmol) was dispersed in DMF (0.8 mL) and THF(3.4 mL), added with pyridine (0.03 mL) and 4-nitrophenylcarbonochloridate (0.07 g, 0.36 mmol), and then stirred for 4 hours.After confirming the completion of the reaction by TLC, n-hexane (3 mL)was added thereto, and stirred for 30 minutes. A solid thus formed waswashed with a mixed solvent of n-hexane:THF=1:1 (12 mL), filtered andthen dried. The dried compound was dispersed in DMF (3 mL), added with2,6-dichloroaniline (0.34 g, 2.08 mmol), and then stirred for 20 minutesunder microwave conditions (250 W, 250 psi, 150° C.). The reactionsolution was diluted with ethyl acetate containing 5% methanol, and thenwashed sequentially with a saturated aqueous solution of NaHCO₃ andwater. The organic layer was dried over anhydrous magnesium sulfate,concentrated, and then purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.03 g, yield: 19%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 9.54 (s, 1H), 9.36 (s, 1H), 8.58 (s,1H), 8.43 (d, J=8.1 Hz, 1H), 7.59 (m, 3H), 7.47 (t, J=8.4 Hz, 1H), 7.32(m, 2H), 7.08 (s, 1H), 4.41 (s, 2H), 2.25 (m, 3H)

LCMS [M+1]: 511

Example 2: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-trifluoromethyl-phenyl)-urea

4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.1 g, 0.31 mmol) was dispersed in DMF (0.8 mL) and THF(3.4 mL), added with pyridine (0.03 mL) and 4-nitrophenylcarbonochloridate (0.07 g, 0.36 mmol), and then stirred for 4 hours.Subsequently, n-hexane (3 mL) was added to the mixture, and stirred for30 minutes. A solid thus formed was washed with a mixed solution ofn-hexane:THF=1:1 (12 mL), filtered and then dried. The dried compoundwas dispersed in DMF (2 mL), added with 2-trifluoromethyl aniline (0.74g, 4.65 mmol), and then stirred for 3 hours at room temperature. Thereaction solution was added sequentially with methanol (6 mL), and asaturated aqueous solution of NaHCO₃, and stirred for 30 minutes. Asolid thus formed was filtered and washed with water. After drying thewashed solid, the solid was purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.07 g, yield: 44%).

¹H-NMR Spectrum(300 MHz, DMSO-d₆): 9.54 (s, 1H), 9.36 (s, 1H), 8.58 (s,1H), 8.43 (d, J=8.1 Hz, 1H), 7.59 (m, 3H), 7.47 (t, J=8.4 Hz, 1H), 7.32(m, 2H), 7.08 (s, 1H), 4.41 (s, 2H), 2.25 (m, 3H)

LCMS [M+1]: 510.0

Example 3: Preparation of1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

2,6-difluoro-benzoic acid (0.04 g, 0.248 mmol) was dispersed in diethylether (3 mL), slowly added with phosphorus pentachloride (PCl₅, 0.057 g,0.273 mmol), and then stirred for 1 hour. Upon completion of thereaction, the organic solvent was concentrated under reduced pressurebelow room temperature, and then the reaction solution was diluted byadding acetone (2 mL). Subsequently, sodium azide (NaN₃, 0.019 g, 0.298mmol) dissolved in water (0.2 mL) was slowly added to the reactionsolution dropwise at 0° C. After stirring the reaction solution for 2hours at room temperature, 2,6-difluoro-benzoyl azide thus formed wasdiluted with ethyl acetate, and then washed with water. The organiclayer was dried over anhydrous magnesium sulfate, dispersed in THF (1mL), added with THF (4 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.04 g, 0.124 mmol), and then stirred for 4 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and then purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.025 g, yield: 42%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.36 (m, 1H), 9.40-9.36 (m,2H), 8.42 (d, J=8.1 Hz, 1H), 8.33 (s, 1H), 7.62-7.57 (m, 2H), 7.48 (t,J=8.4 Hz, 1H), 7.37-7.26 (m, 2H), 7.20-6.82 (m, 3H), 4.40 (s, 2H),2.29-2.19 (m, 3H)

LCMS [M+1]: 478.4

Example 4: Preparation of1-(2-chloro-6-fluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

2-chloro-6-fluorobenzoic acid (0.054 g, 0.31 mmol) was dispersed indiethyl ether (3 mL), slowly added with phosphorus pentachloride (PCl₅,0.074 g, 0.357 mmol), and then stirred for 1 hour. Upon completion ofthe reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (2 mL). Subsequently, sodium azide (NaN₃,0.024 g, 0.372 mmol) dissolved in water (0.2 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 2 hours at room temperature, the reaction solution was diluted withethyl acetate and washed with water. The organic layer was dried overanhydrous magnesium sulfate, dispersed in THF (1 mL), added with THF (4mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.05 g, 0.155 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and then purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.029 g, yield 42%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.35 (m, 1H), 9.40-9.35 (m,2H), 8.42 (d, J=8.1 Hz, 1H), 8.33 (s, 1H), 7.63-7.58 (m, 2H), 7.47 (t,J=8.4 Hz, 1H), 7.41-7.26 (m, 4H), 7.07-6.82 (m, 1H), 4.40 (s, 2H),2.30-2.20 (m, 3H)

LCMS [M+1]: 494.4

Example 5: Preparation of1-(2,6-bis-trifluoromethyl-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

2,6-bis-trifluoromethylbenzoic acid (0.088 g, 0.31 mmol) was dispersedin diethyl ether (3 mL), slowly added with phosphorus pentachloride(PCl₅, 0.068 g, 0.326 mmol), and then stirred for 1 hour. Uponcompletion of the reaction, the organic solvent was concentrated underreduced pressure below room temperature, and then the reaction solutionwas diluted by adding acetone (2 mL). Subsequently, sodium azide (NaN₃,0.024 g, 0.372 mmol) dissolved in water (0.2 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 2 hours at room temperature, 2,6-bis-trifluoromethylbenzoyl azidethus formed was diluted with ethyl acetate, and then washed with water.The organic layer was dried over anhydrous magnesium sulfate, dispersedin THF (1 mL), added with THF (4 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.05 g, 0.155 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and the purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.018 g, yield: 20%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 8.40 (d, J=8.4 Hz, 1H), 8.09-8.06(m, 2H), 7.76 (t, J=8.1 Hz, 1H), 7.63 (d, J=8.1 Hz, 1H), 7.54 (d, J=12.9Hz, 1H), 7.38 (t, J=8.4 Hz, 1H), 7.24 (d/d, J=8.4 Hz, 1H), 6.94 (s, 1H),4.43 (s, 2H), 2.33 (s, 3H)

LCMS [M+1]: 578.4

Example 6: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-fluoro-6-trifluoromethyl-phenyl)-urea

2-fluoro-6-trifluoromethylbenzoic acid (0.058 g, 0.279 mmol) wasdispersed in diethyl ether (3 mL), slowly added with phosphoruspentachloride (PCl₅, 0.064 g, 0.307 mmol), and then stirred for 1 hour.Upon completion of the reaction, the organic solvent was concentratedunder reduced pressure below room temperature, and then the reactionsolution was diluted by adding acetone (2 mL). Subsequently, sodiumazide (NaN₃, 0.024 g, 0.363 mmol) dissolved in water (0.2 mL) was slowlyadded to the reaction solution dropwise 0° C. After stirring thereaction solution for 2 hours at room temperature, the reaction solutionwas diluted with ethyl acetate, and then washed with water. The organiclayer was dried over anhydrous magnesium sulfate, dispersed in THF (1mL), and then introduced to a flask which contained4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.045 g, 0.14 mmol) diluted in THF (4 mL), followed bystirring for 4 hours at 90° C. Upon completion of the reaction, thesolvent was concentrated under reduced pressure, and then purified bysilica gel column chromatography (methylene chloride:methanol=20:1) toobtain the title compound (0.023 g, yield: 32%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 8.41-8.39 (m, 1H), 7.64-7.50 (m,5H), 7.39 (t, J=8.4 Hz, 1H), 7.25 (m, J=8.4 Hz, 1H), 6.94 (s, 1H), 4.43(s, 2H), 2.33 (s, 3H)

LCMS [M+1]: 528.4

Example 7: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea

2,4,6-trifluorobenzoic acid (0.08 g, 0.45 mmol) was dispersed in diethylether (5.7 mL), slowly added with phosphorus pentachloride (PCl₅, 0.11g, 0.52 mmol), and then stirred for 1 hour. Upon completion of thereaction, the organic solvent was concentrated under reduced pressurebelow room temperature, and then the reaction solution was diluted byadding acetone (3.8 mL). Subsequently, sodium azide (NaN₃, 0.035 g,0.545 mmol) dissolved in water (0.28 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring for 2 hours at roomtemperature, 2,4,6-trifluorobenzoyl azide thus formed was diluted withethyl acetate, and then washed with water. The organic layer was driedover anhydrous magnesium sulfate, dispersed in THF (2 mL), added withTHF (7.5 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.073 g, 0.23 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and then purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.026 g, yield: 23%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.46-14.37 (m 1H), 9.47-9.45 (br m,1H), 9.37 (s, 1H), 8.45 (d, J=1.8 Hz, 1H), 8.30-8.27 (br m, 1H),7.63-7.46 (m, 3H), 7.31-7.26 (m, 3H), 7.09-6.84 (m, 1H), 4.42 (s, 2H),2.31-2.21 (m, 3H)

LCMS [M+1]: 496.3

Example 8: Preparation of1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-1-methyl-urea

4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.1 g, 0.31 mmol) was dispersed in a mixed solution of DMF(0.8 mL) and THF (4 mL), and then pyridine (0.05 ml) and 4-nitrophenylcarbonochloridate (0.07 g, 0.36 mmol) were added thereto, followed bystirring for 4 hours. Upon completion of the reaction, n-hexane (3 mL)was added to the resulting a mixture, followed by stirring for 30minutes. A solid thus formed was washed with a mixed solvent ofn-hexane:THF=1:1 (12 mL), filtered, and then dried. The dried compoundwas dispersed in DMF (4 mL), added with 2,6-difluoro-methylaniline(0.294 g, 2.05 mmol), and then stirred for 12 hours at 100° C. Thereaction solution was cooled to room temperature, diluted with ethylacetate containing 5% methanol, and washed with a saturated aqueoussolution of NaHCO₃ and water. The organic layer was dried over anhydrousmagnesium sulfate, filtered, and concentrated. Finally the organic layerwas purified by silica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.018 g, yield:18%). ¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.36 (m, 1H), 9.36 (s,1H), 8.91 (s, 1H), 8.42 (d, J=8.1 Hz, 1H), 7.61-7.34 (m, 5H), 7.26-7.21(m, 2H), 7.07-6.82 (m, 1H), 4.40 (s, 2H), 3.20 (s, 3H), 2.30-2.20 (m,3H)

LCMS [M+1]: 492.4

Example 9: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-pentafluorophenyl-urea

Pentafluorobenzoic acid (0.066 g, 0.31 mmol) was dispersed in diethylether (3 mL), slowly added with phosphorus pentachloride (PCl₅, 0.071 g,0.341 mmol), and then stirred for 40 minutes. Upon completion of thereaction, the organic solvent was concentrated under reduced pressurebelow room temperature, and then the reaction solution was diluted byadding acetone (3 mL). Subsequently, sodium azide (NaN₃, 0.026 g, 0.403mmol) dissolved in water (0.2 mL) was slowly added to the reactionsolution dropwise at 0° C. After stirring the reaction solution for 1hour at room temperature, the reaction solution was diluted with ethylacetate, and then washed with water. The organic layer was dried overanhydrous magnesium sulfate, dispersed in THF (1 mL), and thenintroduced to a flask which contained4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.050 g, 0.155 mmol) diluted in THF (3 mL), followed bystirring for 3 hours at 90° C. Upon completion of the reaction, thesolvent was concentrated under reduced pressure, and then purified bysilica gel column chromatography (methylene chloride:methanol=20:1) toobtain the title compound (0.023 g, yield: 28%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.36 (m, 1H), 9.60 (s, 1H),9.36 (s, 1H), 8.83 (br s, 1H), 8.43 (d, J=8.1 Hz, 1H), 7.62-7.57 (m,2H), 7.50 (t, J=8.4 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.08-6.83 (m, 1H),4.40 (s, 2H), 2.30-2.20 (m, 3H)

LCMS [M+1]: 532.4

Example 10: Preparation of1-(2,5-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea

2,5-difluorobenzoic acid (0.05 g, 0.32 mmol) was dispersed in THF (4mL), and then triethylamine (0.088 mL, 0.63 mmol) anddiphenylphosphorazidate (DPPA, 0.08 mL, 0.36 mmol) were added thereto,followed by stirring for 2 hours at room temperature. After checkingthat 2,5-difluorobenzoyl azide was formed, Compound D (0.051 g, 0.16mmol) was added thereto, followed by stirring for 4 hours at 90° C. Uponcompletion of the reaction, the reaction solution was diluted with ethylacetate containing 5% methanol, and then washed with a saturated aqueoussolution of NaHCO₃. Subsequently, the organic layer was dried overanhydrous magnesium sulfate, and then concentrated under reducedpressure. The concentrate thus obtained was purified by silica gelcolumn chromatography (eluent: methylene chloride:methanol=20:1) toobtain the title compound (0.026 g, yield: 34%).

¹H-NMR Spectrum(300 MHz, DMSO-d₆): 14.47-14.37 (m, 1H), 9.58 (s, 1H),9.37 (s, 1H), 8.96 (s, 1H), 8.45 (d, J=8.4 Hz, 1H), 8.06-7.99 (m, 1H),7.67-7.49 (m, 3H), 7.36-7.09 (m, 2H), 6.89-6.84 (m, 1H), 4.43 (s, 2H),2.31-2.22 (m, 3H)

LCMS [M+1]: 478.4

Example 11: Preparation of1-(2,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

2,4-difluorobenzoic acid (0.04 g, 0.248 mmol) was dispersed in THF (3mL), and then triethylamine (0.069 mL, 0.496 mmol) anddiphenylphosphorazidate (DPPA, 0.075 g, 0.273 mmol) were added thereto,followed by stirring for 2 hours at room temperature.4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.040 g, 0.124 mmol) was added to the mixture, followed bystirring for 4 hours at 90° C. Upon completion of the reaction, thereaction solution was diluted with ethyl acetate containing 5% methanol,and then washed with water and a saturated aqueous solution of NaHCO₃.Subsequently, the organic layer was dried over anhydrous magnesiumsulfate, and then concentrated under reduced pressure. The concentratethus obtained was purified by silica gel column chromatography (eluent:methylene chloride:methanol=20:1) to obtain the title compound (0.024 g,yield: 42%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.36 (m, 1H), 9.39-9.35 (m,2H), 8.64 (s, 1H), 8.43 (d, J=8.1 Hz, 1H), 8.09-8.00 (m, 1H), 7.65-7.59(m, 2H), 7.49 (t, J=8.4 Hz, 1H), 7.35-7.21 (m, 2H), 7.08-6.83 (m, 2H),4.41 (s, 2H), 2.30-2.20 (m, 3H)

LCMS [M+1]: 478.4

Example 12: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,6-trifluoro-phenyl)-urea

2,3,6-trifluorobenzoic acid (0.044 g, 0.248 mmol) was dispersed indiethyl ether (4 mL), slowly added with phosphorus pentachloride (PCl₅,0.057 g, 0.273 mmol), and then stirred for 40 minutes. Upon completionof the reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (3 mL). Subsequently, sodium azide (NaN₃,0.021 g, 0.322 mmol) in water (0.2 mL) was slowly added to the reactionsolution dropwise at 0° C. After stirring the reaction solution for 1hour at room temperature, the reaction solution was diluted with ethylacetate, and then washed with water. The organic layer was dried overanhydrous magnesium sulfate, dispersed in THF (1 mL), and thenintroduced to a flask which contained4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.040 g, 0.124 mmol) diluted in THF (3 mL), followed bystirring for 6 hours at 90° C. Upon completion of the reaction, thesolvent was concentrated under reduced pressure, and then purified bysilica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.022 g, yield:36%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.35 (m, 1H), 9.51 (br s,1H), 9.35 (s, 1H), 8.62 (br s, 1H), 8.43 (d, J=8.1 Hz, 1H), 7.62-7.57(m, 2H), 7.48 (t, J=8.4 Hz, 1H), 7.44-7.34 (m, 1H), 7.31-7.20 (m, 2H),7.07-6.83 (m, 1H), 4.40 (s, 2H), 2.30-2.20 (m, 3H)

LCMS [M+1]: 496.4

Example 13: Preparation of1-(3,5-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]phenyl}-urea

3,5-difluorobenzoic acid (0.04 g, 0.248 mmol) was dispersed in THF (4mL), and then triethylamine (0.069 mL, 0.496 mmol) anddiphenylphosphorazidate (DPPA, 0.075 g, 0.273 mmol) were added thereto,followed by stirring for 1 hour at room temperature.4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.040 g, 0.124 mmol) was added to the reaction solution,followed by stirring for 4 hours at 90° C. The reaction solution wascooled to room temperature, diluted with ethyl acetate, and then washedwater and a saturated aqueous solution of NaHCO₃. The organic layer wasdried over anhydrous magnesium sulfate, and then concentrated underreduced pressure. The concentrate thus obtained was purified by silicagel column chromatography (eluent: methylene chloride:methanol=20:1) toobtain the title compound (0.032 g, yield: 55%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.45-14.36 (m, 1H), 9.50 (s, 2H),9.36 (s, 1H), 8.43 (d, J=8.1 Hz, 1H), 7.64-7.60 (m, 2H), 7.50 (t, J=8.4Hz, 1H), 7.30-7.20 (m, 3H), 7.08-6.77 (m, 2H), 4.41 (s, 2H), 2.30-2.20(m, 3H)

LCMS [M+1]: 478.3

Example 14: Preparation of1-(3,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

3,4-difluorobenzoic acid (0.05 g, 0.32 mmol) was dispersed in THF (4mL), and then triethylamine (0.088 mL, 0.63 mmol) anddiphenylphosphorazidate (DPPA, 0.08 mL, 0.36 mmol) were added thereto,followed by stirring for 2 hours at room temperature. After checkingthat 3,4-difluorobenzoyl azide was formed,4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.051 g, 0.16 mmol) was added thereto, followed by stirringfor 4 hours at 90° C. Upon completion of the reaction, the reactionsolution was diluted with ethyl acetate containing 5% methanol, and thenwashed with a saturated aqueous solution of NaHCO₃. Subsequently, theorganic layer was dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The concentrate thus obtained waspurified by silica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.035 g, yield:46%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.40 (br s, 1H), 9.37 (s, 1H),9.13-9.03 (m, 2H), 8.44 (d, J=8.1 Hz, 1H), 7.71-7.47 (m, 4H), 7.41-7.27(m, 3H), 7.18-7.16 (m, 1H), 7.00 (s, 1H), 4.43 (s, 2H), 2.26 (s, 3H)

LCMS [M+1]: 478.4

Example 15: Preparation of1-(4-cyano-3-fluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea

4-cyano-3-fluorobenzoic acid (0.08 g, 0.48 mmol) was dispersed in THF(6.1 mL), added with triethylamine (0.14 mL, 0.97 mmol) anddiphenylphosphorazidate (DPPA, 0.12 mL, 0.56 mmol), and then stirred for2 hours at room temperature. After checking that 4-cyano-3-fluorobenzoylazide was formed,4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.078 g, 0.24 mmol) was added thereto, followed by stirringfor 4 hours at 90° C. Upon completion of the reaction, the reactionsolution was diluted with ethyl acetate containing 5% methanol, and thenwashed with a saturated aqueous solution of NaHCO₃. Subsequently, theorganic layer was dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The concentrate thus obtained waspurified by silica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.012 g, yield:10%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.40 (br s, 1H), 9.89 (s, 1H), 9.64(s, 1H), 9.37 (s, 1H), 8.44 (d, J=8.7 Hz, 1H), 7.94-7.15 (m, 7H),7.01-6.99 (m, 1H), 4.43 (s, 2H), 2.26 (s, 3H)

LCMS [M+1]: 485.4

Example 16: Preparation of1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)pheny)urea

4-chloro-3-trifluoromethylbenzoic acid (0.08 g, 0.35 mmol) was dispersedin THF (4.5 mL), and then triethylamine (0.1 mL, 0.71 mmol) anddiphenylphosphorazidate (DPPA, 0.09 mL, 0.41 mmol) were added thereto,followed by stirring for 2 hours at room temperature. After checkingthat 4-chloro-3-trifluoromethylbenzoyl azide was formed,4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.057 g, 0.18 mmol) was added thereto, followed by stirringfor 4 hours at 90° C. Upon completion of the reaction, the reactionsolution was diluted with ethyl acetate containing 5% methanol, and thenwashed with a saturated aqueous solution of NaHCO₃. Subsequently, theorganic layer was dried over anhydrous magnesium sulfate, and thenconcentrated under reduced pressure. The concentrate thus obtained waspurified by silica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.012 g, yield:12%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.47-14.37 (m, 1H), 9.36-9.32 (m,2H), 9.25 (s, 1H), 8.45 (d, J=8.1 Hz, 1H), 8.12-8.05 (m, 1H), 7.71-7.61(m, 4H), 7.54-7.48 (m, 1H), 7.33-7.29 (m, 1H), 7.09-6.85 (m, 1H), 4.42(s, 2H), 2.32-2.22 (m, 3H)

LCMS [M+1]: 544.3

Example 17: Preparation of1-(3-chloro-2,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)pheny)urea

3-chloro-2,6-difluorobenzoic acid (0.08 g, 0.41 mmol) was dispersed indiethyl ether (5.2 mL), slowly added with phosphorus pentachloride(PCl₅, 0.099 g, 0.48 mmol), and then stirred for 1 hour. Upon completionof the reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (3.5 mL). Subsequently, sodium azide (NaN₃,0.032 g, 0.50 mmol) dissolved in water (0.25 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 2 hours at room temperature, 3-chloro-2,6-difluorobenzoyl azide thusformed was diluted with ethyl acetate, followed by washing with water.The organic layer was dried over anhydrous magnesium sulfate, dispersedin THF (1.6 mL), added with THF (1.6 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.067 g, 0.21 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated, and thenpurified by silica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.017 g, yield:16%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.47-14.38 (m, 1H), 9.49-9.39 (m,2H), 8.53 (s, 1H), 8.44 (d, J=8.1 Hz, 1H), 7.63-7.47 (m, 4H), 7.31-7.24(m, 2H), 7.09-6.84 (m, 1H), 4.42 (s, 2H), 2.31-2.21 (m, 3H)

LCMS [M+1]: 512.3

Example 18: Preparation of1-(2-chloro-3,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea

2-chloro-3,6-difluorobenzoic acid (0.08 g, 0.41 mmol) was dispersed indiethyl ether (5.2 mL), slowly added with phosphorus pentachloride(PCl₅, 0.099 g, 0.48 mmol), and then stirred for 1 hour. Upon completionof the reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (3.5 mL). Subsequently, sodium azide (NaN₃,0.032 g, 0.50 mmol) dissolved in water (0.25 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 2 hours at room temperature, 2-chloro-3,6-difluorobenzoyl azide thusformed was diluted with ethyl acetate, followed by washing with water.The organic layer was dried over anhydrous magnesium sulfate, dispersedin THF (1.6 mL), added with THF (7 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.067 g, 0.21 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and the purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.038 g, yield: 36%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.46-14.37 (m, 1H), 9.51 (s, 1H),9.37 (s, 1H), 8.58 (s, 1H), 8.44 (d, J=8.1 Hz, 1H), 7.63-7.59 (m, 2H),7.52-7.29 (m, 4H), 7.09-6.84 (m, 1H), 4.42 (s, 2H), 2.31-2.21 (m, 3H)

LCMS [M+1]: 512.3

Example 19: Preparation of1-(4-chloro-2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

4-chloro-2,6-difluorobenzoic acid (0.060 g, 0.31 mmol) was dispersed indiethyl ether (4 mL), slowly added with phosphous pentachloride (PCl₅,0.071 g, 0.341 mmol), and then stirred for 40 minutes. Upon completionof the reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (3 mL). Subsequently, sodium azide (NaN₃,0.026 g, 0.403 mmol) dissolved in water (0.2 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 1 hour at room temperature, the reaction solution was diluted withethyl acetate, and then washed with water. The organic layer was driedover anhydrous magnesium sulfate, dispersed in THF (1 mL), and thenintroduced to a flask which contained4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.050 g, 0.155 mmol) dissolved in THF (3 mL), followed bystirring for 4 hours at 90° C. Upon completion of the reaction, thesolvent was concentrated under reduced pressure, and then purified bysilica gel column chromatography (eluent: methylenechloride:methanol=20:1) to obtain the title compound (0.024 g, yield:30%).

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 14.46-14.37 (m, 1H), 9.86 (s, 1H),9.38 (s, 1H), 8.82 (s, 1H), 8.41 (d, J=8.1 Hz, 1H), 7.63-7.59 (m, 2H),7.52-7.29 (m, 4H), 6.97 (s, 1H), 4.42 (s, 2H), 2.21 (s, 3H)

LCMS [M+1]: 512.3

Example 20: Preparation of1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,5,6-tetrafluoro-phenyl)-urea

2,3,5,6-tetrafluorobenzoic acid (0.08 g, 0.41 mmol) was diluted indiethyl ether (5.2 mL), slowly added with phosphorus pentachloride(PCl₅, 0.099 g, 0.48 mmol), and then stirred for 1 hour. Upon completionof the reaction, the organic solvent was concentrated under reducedpressure below room temperature, and then the reaction solution wasdiluted by adding acetone (3.4 mL). Subsequently, sodium azide (NaN₃,0.032 g, 0.50 mmol) dissolved in water (0.25 mL) was slowly added to thereaction solution dropwise at 0° C. After stirring the reaction solutionfor 2 hours at room temperature, 2,3,5,6-tetrafluorobenzoyl azide thusformed was diluted with ethyl acetate, and then washed with water. Theorganic layer was dried over anhydrous magnesium sulfate, dispersed inTHF (1.6 mL), added with THF (7 mL) containing4-(4-amino-2-fluorophenyl)-7-(5-methyl-1H-imidazol-2-yl)isoindolin-1-one(Compound D, 0.066 g, 0.21 mmol), and then stirred for 3 hours at 90° C.Upon completion of the reaction, the solvent was concentrated underreduced pressure, and then purified by silica gel column chromatography(eluent: methylene chloride:methanol=20:1) to obtain the title compound(0.014 g, yield: 13%)

¹H-NMR Spectrum (300 MHz, DMSO-d₆): 8.45 (d, J=8.1 Hz, 1H), 7.69-7.61(m, 2H), 7.48-7.33 (m, 3H), 7.00 (s, 1H), 4.48 (s, 2H), 2.38 (s, 3H)

LCMS [M+1]: 514.3

The compounds obtained in Examples 1 to 20 are represented by thefollowing structural formula, as shown in Table 1 below.

TABLE 1 Compound Name Formula 11-(2,6-dichloro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

2 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-trifluoromethyl-phenyl)-urea

3 1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

4 1-(2-chloro-6-fluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

5 1-(2,6-bis-trifluoromethyl-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}- urea

6 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-fluoro-6-trifluoromethyl- phenyl)-urea

7 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea

8 1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3- dihydro-1H-isoindol-4-yl]-phenyl}-1-methyl-urea

9 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-pentafluorophenyl-urea

10 1-(2,5-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin- 4-yl)phenyl)urea

11 1-(2,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

12 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,6-trifluoro-phenyl)-urea

13 1-(3,5-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

14 1-(3,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea

15 1-(4-cyano-3-fluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1- oxoisoindolin-4-yl)phenyl)urea

16 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea

17 1-(3-chloro-2,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1- oxoisoindolin-4-yl)phenyl)urea

18 1-(2-chloro-3,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1- oxoisoindolin-4-yl)phenyl)urea

19 1-(4-chloro-2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}- urea

20 1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,5,6-tetrafluoro-phenyl)-urea

The compounds prepared from Examples were tested for biological assaysas follows.

Evaluation of biological activities of the compounds in accordance withthe present invention may be conducted by any conventional methods knownin the art. Appropriate test methods are well known in the art. Thefollowing tests are examples for assaying effects of the inventivecompounds on various kinases, which are not limited hereto. Thecompounds of the present invention show their activities in at least oneof the following assays.

Experimental Example 1: Assay for BTK Inhibition Activity (ELISA Method)

In order to evaluate the activity of the compounds of the presentinvention as a BTK inhibitor, commercially available BTK (Promega) wasused for this experiment. Specifically, an enzymatic reaction wasconducted by mixing 0.4 nM of BTK enzyme, 40 μM of biotin-S1 substratepeptide and 50 μM of ATP in a reaction buffer (15 mM Tris-HCl (pH 7.5),20 mM MgCl₂, 2 mM MnCl₂, 2 mM DTT, 0.1 mg/ml BSA). The mixture wastreated with the test compounds at predetermined concentrations andallowed to react for 20 minutes at 30° C. Upon completion of thereaction, the activities of the test compounds were measured by ELISAmethod. The absorbance value of an untreated sample was used as acontrol (100% control). BTK enzyme activities were measured aftertreatment with various concentrations of the test compounds, and theconcentration of test compounds resulting in 50% inhibition of BTKenzyme as compared to the control was determined as IC₅₀ of BTKinhibitor.

BTK inhibition activities of several compounds among the inventivecompounds were randomly tested. The results are shown in Table 2 below.

TABLE 2 Results of BTK inhibition activities of representative compoundsCompound BTK IC₅₀ (nM) 3 1.00 4 0.90 7 0.10 9 0.40 12 0.28 14 5.60 155.00 17 0.05 18 0.07 19 4.00 20 0.90

Experimental Example 2: Histamine Release Assay

According to Kawakami et al., inhibition of BTK activity in mast cellsreduces production of a mediator (e.g., histamine), and a lipidmediator, or secretion of cytokine. (Reference [J Immunol. 2000 Aug. 1;165 (3):1210-9. Redundant and opposing functions of two tyrosinekinases, Btk and Lyn, in mast cell activation. Kawakami Y, Kitaura J,Satterthwaite A B, Kato R M, Asai K, Hartman S E, Maeda-Yamamoto M,Lowell C A, Rawlings D J, Witte O N, Kawakami T]).

Histamine release assays were performed with reference to the methoddisclosed in the article, FEBS Lett. 2002 Sep. 11; 527 (1-3):274-8.Silencing of Bruton's tyrosine kinase (Btk) using short interfering RNAduplexes (siRNA). Heinonen J E, Smith C I, Nore B F, and the amount ofhistamine was measured by an enzyme immunoassay.

The RBL-2H3 cell line, purchased from KCLB (Korean Cell Line Bank), wasgrown in a DMEM medium supplemented with 10% (v/v) FBS at 37° C. in a 5%CO₂ incubator for 72 hours. The cells were transferred into 96-wellplates at a density of 10,000 cells/well, and cultured at 37° C. in the5% CO₂ incubator for 24 hours.

The cells were treated with 500 ng/mL of monoclonal anti-DNP (sigma) andeach of 0.001, 0.01, 0.1, 1.0 and 10 μM of the test compound in 100%(v/v) dimethyl sulfoxide (DMSO). The cells were treated only with 100%(v/v) DMSO, which was used as a control.

The treated samples were cultured at 37° C. in the 5% CO₂ incubator for24 hours. Then, histamine release was measured according tomanufacturer's instructions (EIA histamine kit, immunotech). Each wellwas treated with 50 μL of a histamine release buffer, and allowed toreact for 30 minutes at 37° C. Upon completion of the reaction, 100 μLof a sample from each well was transferred to a new plate, and then wasthoroughly mixed with 25 μL of an acetylation buffer and 25 μL of anacetylation reagent. 50 μL of the acetylation sample thus prepared wastransferred to a plate coated with an antibody, mixed with 200 μL ofhistamine alkaline phosphatase conjugate, and then allowed to react for18 hours at 4° C. Once the reaction is completed, the sample from theplate is removed, and then 200 μL of a wash buffer was added to washthree times. 200 μL of a substrate was added thereto, and the mixturewas allowed to react at room temperature for 30 minutes. The reactionwas terminated by adding 50 μL of a stop solution, and absorbance of thesamples was read at 406 nm using Benchmark Plus (Biorad). The histaminerelease level was calculated based on absorbance of the test groupagainst that of the control group. The EC₅₀ (μM) values, in which testcompounds reduce the histamine release by 50%, were determined by usingMicrosoft Excel graphic program.

In order to evaluate efficacies of the inventive compounds as ananti-inflammatory drug, histamine release tests of several compoundsamong them were randomly conducted. The EC₅₀ values of the compounds aresummarized in Table 3. The results indicate that the compounds obtainedin Examples according to the present invention have excellent efficacy.

TABLE 3 Results of histamine release test of representative compoundsCompound Histamine Release EC₅₀ (μM) 3 0.45 4 0.25 7 0.22 9 0.35 12 0.3014 0.20 15 0.22 17 0.04 18 0.30 19 0.30 20 0.30

Experimental Example 3: MTS Assay Based on Anti-Proliferation Assay

MTS assay was performed to evaluate the anti-proliferative activities ofthe inventive compounds via inhibition on extracellular signal-regulatedkinase (Barltrop, J. A. et al., (1991)5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analog of3-(4,5-dimethylthiazolyl)-2,5,-diphenyltetrazolium bromide (MTT)reducing to purple water soluble formazans as cell-viability indicators.Bioorg. Med. Chem. Lett. 1, 611-4; Cory, A. H. et al., (1991) Use of anaqueous soluble tertrazolium/formazan assay for cell growth assays inculture. Cancer Comm. 3, 207-12).

Human lymphoma cell lines Jeko-1 (ATCC), Mino (ATCC), H9 (Korean CellLine Bank) and SR (ATCC), and human leukemia cell lines MV4-11 (ATCC),Molm-13 (DSMZ) and Ku812 (ATCC) were used for the test according to theprocedure shown below.

Each of Jeko-1, Mino, H9, SR, MV4-11, Molm-13 and Ku812 cells weretransferred into 96-well plates containing RPMI1640 medium (GIBCO,Invitrogen) supplemented with 10% FBS at a density of 10,000 cells/well,and then incubated for 24 hours under conditions of 37° C. and 5% CO₂.The wells were treated with each of 0.2, 1, 5, 25 and 100 μM, of thetest compounds. The well was treated with DMSO in an amount of 0.08 wt%, which is the same amount as in the test compounds, which was used asa control. The resulting cells were incubated for 48 hours.

MTS assays are commercially available and include the Promega CellTiter96® Aqueous Non-Radioactive Cell Proliferation Assay. MTS assays wereperformed in order to evaluate cell viability of the test compounds. 20μL of a mixed solution of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (“MTS”) and phenazine methosulfate (PMS) was added to eachwell, and then incubated for 2 hours at 37° C. Then absorbance of thesamples was read at 490 nm. The anti-proliferation activity level wascalculated based on absorbance of the test compounds against that of theuntreated control group. The EC₅₀ (μM) values, in which test compoundsreduce the growth of cancer cells by 50% were calculated.

An assay for anti-proliferation activity was conducted by using Jeko-1,Mino, H9 and SR lymphoma cells so as to evaluate the effectiveness ofthe inventive compounds as an anti-inflammatory agent as well as ananti-cancer agent. The EC₅₀ values thereof are shown in Table 4 below.

TABLE 4 Anti-proliferation assay of the representative compounds againstlymphoma cells EC₅₀ in EC₅₀ in EC₅₀ in EC₅₀ in Compound Jeko-1 (μM) Mino(μM) H9 (μM) SR (μM) 3 0.10 — 0.03 0.100 4 0.10 — 0.03 0.030 7 0.030.018 0.007 0.015 9 0.03 — 0.006 0.018 12 0.15 — 0.04 0.020 14 0.03 —0.02 0.018 15 0.10 — 0.19 0.150 17 0.03 0.018 0.02 0.025 18 0.03 — 0.020.020 19 0.03 — 0.04 0.020 20 0.03 — 0.01 0.017

Moreover, some inventive compounds that have exhibited excellentefficacies against lymphoma cells were further subjected to ananti-proliferation assay against leukemia cells to confirm theirexcellent efficacies. The EC₅₀ values thereof are shown in Table 5below.

TABLE 5 Anti-proliferation assay of the representative compounds againstleukemia cells EC₅₀ in MV4-11 EC₅₀ in EC₅₀ in Compound (μM) Molm-13 (μM)Ku812 (μM) 7 0.002 0.003 0.600 17 0.004 0.012 0.190

What is claimed is:
 1. A compound selected from the group consisting ofa compound of formula (I), pharmaceutically acceptable salts, esters,prodrugs, hydrates, and solvates thereof:

wherein, A is

and R₈ is hydrogen, halogen, or C₁₋₃alkyl; R₃ and R₇ are eachindependently hydrogen, halogen, cyano, difluoromethyl ortrifluoromethyl, with the proviso that at least two of R₃ to R₇ areindependently halogen, cyano, difluoromethyl or trifluoromethyl.
 2. Thecompound of claim 1, wherein R₃ to R₇ are each independently hydrogen,fluoro, chloro, bromo, iodo, cyano, difluoromethyl or trifluoromethyl.3. The compound of claim 1, wherein R₁ and R₂ are each independentlyhydrogen or methyl; R₃ to R₇ are each independently hydrogen, fluoro,chloro, cyano or trifluoromethyl; and R₈ is hydrogen or fluoro.
 4. Thecompound of claim 1, wherein the compound of formula (I) is selectedfrom the group consisting of: 1)1-(2,6-dichloro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;2)1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;3)1-(2-chloro-6-fluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;4)1-(2,6-bis-trifluoromethyl-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;5)1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2-fluoro-6-trifluoromethyl-phenyl)-urea;6)1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea;7)1-(2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-1-methyl-urea;8)1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-pentafluorophenyl-urea;9)1-(2,5-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;10)1-(2,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;11)1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,6-trifluoro-phenyl)-urea;12)1-(3,5-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;13)1-(3,4-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;14)1-(4-cyano-3-fluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;15)1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;16)1-(3-chloro-2,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;17)1-(2-chloro-3,6-difluorophenyl)-3-(3-fluoro-4-(7-(5-methyl-1H-imidazol-2-yl)-1-oxoisoindolin-4-yl)phenyl)urea;18)1-(4-chloro-2,6-difluoro-phenyl)-3-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-urea;and 19)1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,3,5,6-tetrafluoro-phenyl)-urea.5. A pharmaceutical composition comprising the compound of claim 1 as anactive ingredient for the treatment or relief of one or more diseasescaused by abnormal or uncontrolled activation of protein kinase.
 6. Thepharmaceutical composition of claim 5, wherein said protein kinase isABL, ACK, AXL, Aurora, BLK, BMX, BTK, CDK, CSK, DDR, EPHA, FER, FES,FGFR, FGR, FLT, FRK, FYN, HCK, IRR, ITK, JAK, KDR, KIT, LCK, LYN, MAPK,MER, MET, MINK, MNK, MST, MUSK, PDGFR, PLK, RET, RON, SRC, SRM, TIE,SYK, TNK1, TRK TNIK, or a combination thereof.
 7. The pharmaceuticalcomposition of claim 5, wherein the one or more diseases caused byabnormal or uncontrolled activation of protein kinase are cancer,inflammation associated with rheumatoid arthritis and osteoarthritis,asthma, allergy, atopic dermatitis, or psoriasis, and wherein the canceris selected from one or more of the group consisting of lymphoma,leukemia, blood cancer, stomach cancer, non-small cell lung cancer,liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer,brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis,uterine cancer, cervical cancer, ovarian cancer, head and neck cancer,esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer,sarcoma, prostate cancer, urethral cancer, bladder cancer, fibroadenoma,and glioblastoma.
 8. The pharmaceutical composition of claim 7, whereinthe disease is cancer and the cancer is selected from one or more of thegroup consisting of lymphoma, leukemia, blood cancer, stomach cancer,non-small cell lung cancer, liver cancer, colorectal cancer, small bowelcancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breastcancer, sclerosing adenosis, uterine cancer, cervical cancer, ovariancancer, head and neck cancer, esophageal cancer, thyroid cancer,parathyroid cancer, kidney cancer, sarcoma, prostate cancer, urethralcancer, bladder cancer, fibroadenoma, and glioblastoma.
 9. Thepharmaceutical composition of claim 5, which further comprises at leastone additive selected from the group consisting of antibiotic,alkylating agent, antimetabolite, hormonal agent, immunological agent,interferon-type agent and anticancer agent.
 10. A method for thetreatment or relief of diseases caused by abnormal or uncontrolledactivation of protein kinase in a mammal, comprising administering tothe mammal the compound of claim 1, wherein the diseases caused byabnormal or uncontrolled activation of protein kinase are cancer,inflammation associated with rheumatoid arthritis and osteoarthritis,asthma, allergy, atopic dermatitis, and/or psoriasis, and wherein thecancer is selected from one or more of the group consisting of lymphoma,leukemia, blood cancer, stomach cancer, non-small cell lung cancer,liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer,brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis,uterine cancer, cervical cancer, ovarian cancer, head and neck cancer,esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer,sarcoma, prostate cancer, urethral cancer, bladder cancer, fibroadenoma,and glioblastoma.
 11. A method comprising incorporating the compound ofclaim 1 into a medicament for the treatment or relief of diseases causedby abnormal or uncontrolled of protein kinase, wherein the diseasescaused by abnormal or uncontrolled activation of protein kinase areselected from one or more of the group consisting of cancer,inflammation associated with rheumatoid arthritis and osteoarthritis,asthma, allergy, atopic dermatitis, and/or psoriasis, and wherein thecancer is selected from one or more of the group consisting of lymphoma,leukemia, blood cancer, stomach cancer, non-small cell lung cancer,liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer,brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis,uterine cancer, cervical cancer, ovarian cancer, head and neck cancer,esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer,sarcoma, prostate cancer, urethral cancer, bladder cancer, fibroadenoma,and glioblastoma.
 12. The compound of claim 1, wherein the compound is1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea.13. A pharmaceutical composition comprising at least onepharmaceutically acceptable carrier and as an active ingredient atherapeutically effective amount of at least one compound of claim 1.14. The pharmaceutical composition of claim 13, wherein the compound is1-{3-fluoro-4-[7-(5-methyl-1H-imidazol-2-yl)-1-oxo-2,3-dihydro-1H-isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea.15. The method of claim 10, wherein the disease is cancer, and thecancer is selected from one or more of the group consisting of lymphoma,leukemia, blood cancer, stomach cancer, non-small cell lung cancer,liver cancer, colorectal cancer, small bowel cancer, pancreatic cancer,brain cancer, bone cancer, melanoma, breast cancer, sclerosing adenosis,uterine cancer, cervical cancer, ovarian cancer, head and neck cancer,esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer,sarcoma, prostate cancer, urethral cancer, bladder cancer, fibroadenoma,and glioblastoma.